Amplified fragment length polymorphism diversity of cultivated white oyster mushroom pleurotus ostreatus

HAYATI Journal of Biosciences March 2010
Vol. 17 No. 1, p 21-26
EISSN: 2086-4094

Available online at:
http://journal.ipb.ac.id/index.php/hayati
DOI: 10.4308/hjb.17.1.21

Amplified Fragment Length Polymorphism Diversity of Cultivated
White Oyster Mushroom Pleurotus ostreatus
MUHAMMAD JUSUF
Research Center for Bioresources and Biotechnology, Faculty of Mathematics and Natural Sciences,
Bogor Agricultural University, Darmaga Campus, Bogor 16680, Indonesia
Phone: +62-251-8622833, Fax: +62-251-8621724, E-mail: m_jusuf@ipb.ac.id
Received November 30, 2009/Accepted February 17, 2010
Amplified fragment length Polymorphism (AFLP) analysis was performed to study the genetic diversity of
fifteen isolates of white oyster mushroom (Pleurotus ostreatus) originated from different places in Indonesia
(Java, Sumatra, Bali, and Kalimantan) and Thailand. The majority of studied isolates originated from commercial
mushroom, and some of them were collected from nature. AFLP analysis revealed 202 loci of positive DNA bands.
Heterozygosity value showed polymorphisms of the fifteen studied isolates. Correlation and cluster analyses of
the isolates showed that isolate from Thailand (BNK isolate), isolate from Bogor (AMD isolate), and isolate from

Purwokerto (USX isolate) were distinctly different from the other isolates indicated by positive bands with DNA
size greater than 650 bp. Isolate from Kalimantan (BJM isolate) was distinctly different from Java island
isolates. Generally there was no correlation of AFLP variability and geographic distribution of commercial strain
isolates.
Key words: Plerotus ostreatus, biodiversity, AFLP, cluster analysis, breeding
___________________________________________________________________________

INTRODUCTION
White oyster mushroom (Pleurotus ostreatus) is an
important edible and cultivated mushroom, and it was the
second most important mushroom in the world market,
after button mushroom (Agaricus bisporus) (Chang 1999).
In Indonesia this mushroom was cultivated mushroom by
either traditional or modern farming, and the market was
very promising. Mushroom cultivation became one
alternative of agribusiness in Indonesia. In nature, P.
ostreatus and other Pleurotus sp. live on dead wood as
saprobe primary decomposer. Their ability to produce
lignin degrading enzymes (Velazquez-Cedeno et al. 2004;
Widiastuti et al. 2008), make them possible to be used as

lignocellulosic waste degrading agents.
Despite of economical prospective it is very limited
information of their genetics and breeding to support
development of mushroom cultivation in Indonesia.
Farmers usually used mushroom spawn received from the
spawn producers without ability to select quality of the
spawn. Thus a study on genetics and breeding of this
mushroom is very important for their cultivation.
Construction and evaluation of a germplasm or genetic
resouces is the first important step of breeding program.
The collected materials could be natural or commercial
isolates. The purpose of the collection is to build a genetic
diversity as foundation of genetic improvement program.
White oyster mushroom is propagated somatically, and
majority of farmers use spawn produced from the same
spawn poducer. This practical culture can lead to a genetic
homogenity of cultivated mushroom in a region. In order
to build a genetic diversity of the mushroom, genetic

materials have to be collected from several locations, and

a thourough evaluation have to be performed to analyse
the genetic variability.
This paper reported the genetic evaluation of the
mushroom analysed by amplified fragment length
polymorphism (AFLP) technique (Vos et al. 1999). The
technique was frequently used to study biodiversity of
many organisms such as papaya (Kim et al. 2002),
sunflower (Al-Chaarani et al. 2004), tomato (Park et al.
2004), and garlic (Volk et al. 2004).
MATERIALS AND METHODS
Genetic Materials. Fifteen white oyster mushroom
isolates used in this work were isolated from commercial
mushroom or laboratory stocks collected from different
places in Indonesia (Java, Sumatera, Kalimantan, and Bali)
and Thailand (Table 1). The isolates were kept on slant
agar of potato dextrose agar (PDA) media supplemented
with 500 mg chloramphenicol.
DNA Extration. DNA was extracted from either mycelia
or from fruiting bodies. The mycelia was produced on
liquid peptone yeast extract sugar media (Eger 1976). The

mycelia was harvested in a sieve after two weeks of
incubation and squeezed between filter papers to remove
liquid as much as possible. The pre-dried mycelia was
peeled off from the filter paper, folded, and immersed into
liquid nitrogen.
A total of 0.1 g mycelia or fruiting bodies from each
isolate was immersed into liquid nitrogen, grounded
thoroughly on mortar and then transferred into universal
bottles containing 3 ml extraction buffer. The bottles were

22

JUSUF

gently agitated and incubated at 65 oC for 35 minutes.
DNA was then extracted using a procedure of Sambrook
et al. (1989) with an extraction buffer of Reader and Broda
(1985). The extracted DNA pellet was resuspended with
50 µl H2O, and RNase was added and incubated overnight
at 37 oC. RNase was inactivated by heating at 70 oC for

10 minutes. The purity and quality of DNA was verified
using UV spectrophotometer and electrophoresis.
AFLP Template Preparation. DNA template was
employed using restriction enzymes of PstI and MseI
AFLP adapters. A total 250-500 ng genomic DNA was
digested by 5 U PstI and 5 U MseI restriction enzymes at
37 oC for 3 h in NE Buffer (10x) provided by Biolabs Inc.
Then 10 mM ATP was added to the solution. Adapters
were prepared by adding top and bottom strands with
proportion of 3.4 µl Mtop: 4.17 µl Mbot (50 pMol/µl MSeI
adapter), and 0.67 µl Ptop: 0.42 µl Pbot (5 pMol/µl PstI
adapter). Ligation was performed by adding 3 U/µl of T4
DNA ligase, then the mixture was diluted up to 10 times
using TE buffer (pH 8.0) and stored at -20 oC.
AFLP Fragments Amplification. Amplification process
was conducted in two steps. The first step was preamplification to produce AFLP template. And the second
step was selective amplification to produce specific
fragments that will be appeared on electrophoresis gel.
The AFLP reaction employed PstI and MseI primers which
were produced by Biolegio BV. The AFLP primers for preamplification had no selective nucleotides; the sequence

of PstI primer (P00) was 5’GACTGCGTACATGCAG3’ and
MSeI primer (M02) was 5’GATGAGTCCTGAGTAAC3’.
The primers for selective amplification had two additional
selective nucleotides, i.e. PstI primer (P11) sequence
(5’GACTGCGTACATGCAGAA3’), and MSeI primer
(M50) sequence (5’GATGAGTCCTGAGTAAACAT3’).
The P11 primer was labeled with IRDye 700.
Preamplification using PCR PTC-100TM MJ apparatus
was set up for 24 cycles; each cycle consisted of 30
seconds of DNA denaturing (94 oC), 30 seconds of primers
annealing (56 oC), and 60 seconds of extension (72 oC). A
total of 5 µl RL mixture was added with 0.6 µl (30 ng/µl) Pst
I primer (P00), 0.6 µl (30 ng/µl) MSeI primer (M02), 0.4 ìl
dNTP 10 mM, 2 ìl PCR buffer 10 time concentration,
0.08 ìl (5 unit/ìl) super Taq and 11.32 ìl ddH2O. Quality
and quantity of preamplification product was verified by
using electrophoresis.
Selective amplification had the same procedure as
preamplification, except for DNA templates and AFLP
primers. The product of preamplification was diluted 5

times, then 5 µl of it was taken as DNA template for selective
amplification. The primers used in this step were M50 and
P11 primers.
Gel Preparation and Electrophoresis. Electrophoresis
of DNA fragments was done using LI-COR 4300 DNA
Analyzer apparatus. A plate of 6.5% polyacrylamide gel
was made by mixing 23 ml ready to use solution of KB
plus (LI COR Inc), 15 µl TEMED and 150 µl of 10%
ammonium persulphate. The plate was then fixed on
electrophoresis apparatus, and added with TBE buffer
(10 X). Pre-electrophoresis was performed for 30 minutes

HAYATI J Biosci

with 20 watt power to increase temperature up to 50 oC.
Before loading the DNA samples and DNA standard
marker was denaturated at 90 oC for 3 minutes and put
on ice.
Data Analysis. LI-COR 4300 DNA Analyzer provided,
as output, an image of DNA fragment bands of

electrophoretic gel. The bands was then interpreted and
scored using Saga Automated AFLP Software; the
presence of band was scored as one (1) and the absence
was scored as zero (0), and the data were transfered to a
SPSS datasheet to be determined of their genetic
heterozygosity (Nei 1973), correlation and clustering
analysis (Mardia et al. 1979).
RESULTS
AFLP Loci and Polymorphisms. AFLP analysis of
fifteen white oyster mushroom isolates has revealed 202
loci differentiated by size of DNA bands consisting of 42
loci with DNA fragment size greater than 650 bp, 60 loci in
the range of 350-650 bp, and 100 loci in the range of 50350 bp (Figure 1, Table1). The pragment size greater than
650 bp was possessed by four isolates i.e. BNK, USX,
AMD and BJM isolates. The DNA fragment size of 350650 bp was possessed by all isolates, however it was still
dominated by the four isolates mentioned previously. The
last DNA fragment of 50-350 bp was appeared almost
similar on all isolates. Total mean value of heterozygosity
was 0.31, signifying the genetic polymorphisms of the
fifteen isolates.

Correlation and Cluster Analyses. Correlation
analysis generated several correlating functions of the
fifteen isolates and 202 AFLP loci. Those correlations
could be presented by similar projection coordinates of
the isolates and loci on a same correlation plane spanned
by correlation axis (Figure 2). The correlation plane was
divided into two correlation axis and four quadrants
namely as northwest, northeast, southeast, and
southwest. AMD, BNK, and USX isolates were separated
from the other thirteen isolates, the BNK isolate was on
northwest quadrant, and AMD and USX isolates were on
southwest quadrant. On these quadrants, they were
projected mostly the loci with DNA bands greater than
450 bp for the 1st up to 80th loci. The other isolates were
mostly concentrated on northeast quadrant which were
corresponded to the loci with DNA size of 50-450 bp.
On clustering dendogram of fifteen isolates (Figure 3)
it was seen that BNK isolate was separated from the others
with 25% dissimilarity, AMD isolate was separated with
17% dissimilarity, USX isolate was separated with 10%

dissimilarity, and BJM isolate was separated with 6%
dissimilarity. The rest of eleven isolates consisted of the
isolates in northeast quadrant (Figure 2) were gathered in
one group.
To see better diversity among the twelve isolates
instead of BNK, AMD, and USX isolates, it was performed
the second correlation and clustering analyses for those
twelve isolates and 130 loci possessed by the isolates
(Figure 4 & 5). BJM isolate from Kalimantan was took a

Vol. 17, 2010

Mushroom Diversity

a

b

23

c

Figure 1. AFLP gel electrophoresis of three groups based on DNA sizes (a) >= 650 bp, (b) 350 bp-650 bp, (c) 50 bp – 350 bp. The number
indicated labels of (0) DNA molecular weight standard, (1) BNK,(2) USX, (3) CBB, (4) PGN, (5) TBN, (6) CLM, (7) WRM,
(8) LIP, (9) AMD, (10) KRT, (11) BBR, (12) MTR, (13) CSR, (14) USO, and (15) BJM isolates.

Table 1. Number of AFLP loci and the origins of fifteen isolates of white oyster mushroom (Pleurotus ostreatus)
Isolates
Total existing loci
BNK
USX
CBB
PGN
TBN
CLM
WRM
LIP
AMD
KRT
BBR
MTR
CSR
USO
BJM
Heterozygosity

Origin

Supermarket, Bangkok
UNSOED Purwokerto
Traditional market Cibeber, Cianjur
Spawn grower Pagentongan, Bogor
Supermarket, Tabanan, Bali
Farmer Cileumeuh, Cilacap
Farmer Wiramastra Banjarnegara
LIPI, Bogor
Farmer Bogor
Farmer Kerataon, Magetan
Farmer Bibrik, Madiun
Supermarket Metro-Lampung
Spawn grower Cisarua, Sukabumi
UNSOED, Perwokerto
Traditional market, Banjarmasin.

DNA fragments size and number of positive loci
>= 650 bp
350-650 bp
50-350 bp
42
40
6
0
0
0
0
0
0
27
0
0
0
0
0
2
0.21 ± 0.029

position on southeast quadrant separated from the other
isolates which majority came from Java Island. The BJM
isolate corresponded to the loci with 350-450 bp band size
of the 70th to 102nd loci. Two other isolates, CBB and PGN
isolates, took places on positive coordinate of dimension

59
41
35
18
12
9
5
2
6
40
9
3
2
4
8
23
0.30 ± 0.012

101
10
52
77
73
75
69
70
50
53
60
55
44
48
53
61
0.35 ± 0.010

Total
202
91
93
95
85
84
74
72
56
120
69
58
46
52
61
86
0.31 ± 0.010

one, while the rests of isolates occupied on negative part
of dimension one. On the dimension two, BBR, MTR, and
USO isolates was in southwest quadrant separated from
LIP, BBR, WRM, CLM, KRT, and TBN isolates which were
in northwest quadrant.

24

JUSUF

HAYATI J Biosci

On dendogram, it was seen that BJM isolate was
separated from others with 25% dissimilarity, CBB and
a

2

PGN isolates were separated with 11 and 10% dissimilarity
respectively, KRT, and LIP isolates had 6% dissimilarity
(Figure 5). The seven isolates were separated into three
groups i.e. firstly the group of MTR and CSR isolates,
secondly the group of CLM, WRM, BBR, and TBN
isolates, and thirdly the group of USO isolate.
a

1.5

1.0

0
Dimension 2

Dimension 2

1

-1

0.0
-0.5

-2
-3
b

0.5

-2

-1
Dimension 1

0

1
-1.0

4
-1.5
-1.0

-0.5

0.0
0.5
1.0
Dimension 1

2

1.5

2.0

Dimension 2

b

0
Dimension 2

3

-1

2
1
0

-4
-4

-1
-3

-2

-1
0
1
Dimension 1

-2

Figure 2. Projection of (a) fifteen isolates and (b) 202 loci on
correlation plane. The number indicated labels of (1)
BNK, (2) USX,(3) CBB,(4) PGN, (5) TBN, (6) CLM,
(7) WRM, (8) LIP, (9) AMD, (10) KRT, (11) BBR, (12)
MTR, (13) CSR, (14) USO, and (15) BJM isolates.
0

5

10

15

20

25

MTR
CSR
USO
CLM
WRM
BBR
TBN
KRT
LIP
PGN
CBB
BJM
USX
AMD
BNK
Figure 3. Dendogram of clustering analysis of fifteen isolates of
white oyster mushrooms.

-3
-2

2
4
Dimension 1
Figure 4. Projection of (a) tweleve isolates and (b) 130 AFLP loci
on correlation plane. The number indicated labels of
(4) PGN, (5) TBN, (6) CLM, (7) WRM, (8) LIP, (9)
CBB, (10) KRT, (11) BBR, (12) MTR, (13) CSR, (14)
USO, and (15) BJM isolates.
0

0

5

10

15

20

25

MTR
CSR
USO
CLM
WRM
BBR
TBN
KRT
LIP
PGN
CBB
BJM
Figure 5. Dendogram of clustering analysis of tweleve isolates of
white oyster mushrooms.

Vol. 17, 2010

Mushroom Diversity

DISCUSSION
AFLP of the fifteen isolates had revealed 202 loci of
positive DNA bands, and the average value of
heterozygosity of those loci was 0.31, which was signified
the polymorphisms of the isolates. This information
indicated that strategy to collect genetic materials from
many locations resulted a collection of polymorphic
genetic resources.
Correlation and clustering analyses based on 202 AFLP
loci showed that BNK, AMD, and USX isolates were
distinctly different from the other isolates. The BNK isolate
originated from Thailand had 40 loci from a total of 42 loci
greater than 650 bp. The loci were not possessed by the
majority of Indonesian isolates. In the contrary, BNK
isolate had only 10 loci from a total of 100 loci with DNA
bands size of 50-350 bp which was possessed by the
majority of Indonesian isolates.
AMD isolate received from a farmer in Bogor had a
high degree of dissimilarity to the other Indonesian
isolates. This isolate was originally from IPB (Bogor
Agricultural University) strain (Triyatno, personal
communication); and this strain was possibly the isolate
which was originally introduced from Thailand (Sudirman,
personal communication). Similarly to BNK isolate, this
isolate had a number of loci with DNA bands greater than
650 bp, that was not possessed by other Indonesian
isolates. This result showed genetic differences between
Thailand origin mushroom isolates (BNK and AMD
isolates) and Indonesian mushroom isolates. Other
laboratory experiments showed that AMD and BNK
isolates had a high similarity in quantitative traits, and
they were different from Indonesian isolates. AMD and
BNK isolates had higher biomass production than that of
Indonesian isolates (unpublished data).
USX isolate had the third highest differences from the
majority of studied isolates. The USX and USO isolates
are the strains isolated from Indonesian indigenous
mushrooms. The AFLP analysis showed the differences
between those two isolates. the USX isolate had different
fruiting bodies texture than that of USO isolate (Mumpuni
2007, personal communication). It was suggested that USX
isolate belonged to Pleurotus sp., but it was not the
species of P. osteratus. This AFLP data supported that
opinion, however to confirm this indication it is needed
an interfertility test of USX isolate and a P ostreatus strain.
Correlation and clustering analyses of the isolates
instead of BNK, AMD, and USX isolates appeared that
BMJ isolate was very distinct from the other isolates
(Figure 4). The BJM isolate was originally from indigenous
Kalimantan mushroom collected from nature and sold in a
traditional market. Two other isolates isolated from nature
were USO and PGN isolates. Those isolates were originally
from University of General Sudirman (UNSOED)
Purwokerto and Research Center for Forest Product, Bogor,
Indonesia respectively. The BJM, OSU, and PGN isolates
were shown distinctly different on either correlation plane
or clustering dendogram (Figure 4 & 5). The dissimilarity
between those three indigenous isolates can be interpreted
as the diversity of three regional origins.

25

The other isolates were originaly from commercial
strains. Separation or regrouping of those isolates did
not correspond to the geographical distribution. For
instance MTR and TBN isolates from cultivated
mushrooms collected from Metro Lampung, and Tabanan
Bali respectively, were not distinctly separated from Java
isolates, i.e BJM isolate (Figure 5). This fact could be
caused by inter region exchange of mushroom spawn,
because many mushroom cultivators outside of Java
Island looked for mushroom spawn in Java (Parlindungan
2003).
In summary, the effort to collect genetic materials from
different locations resulted a high diversity of polymorphic
genetic resources. This result is very important as basic
data for future breeding program. And interfertility
experiments can be performed between genetically
different isolates. AMD and BNK isolates that were very
different from the other isolates can be used as genetic
sources to improve Indonesian indigenous strains of white
oyster mushroom.
ACKNOWLEDGEMENT
This research was supported by Hibah Bersaing
research grant of Directorate General of Higher Education,
Department of Education, Indonesia. The author thanks
to Bogor Agricultural University and Directorate General
of Higher Education for the awarded grant. We also thank
to Aris Mumpuni from University of General Soedirman,
Indonesia for providing several mushroom isolates used
in this research, and Lisdar I. Sudirman for scientific
discussion. We also thank to Elvavina, Sri Maria Ulfa,
and Puspiari for DNA preparation and LI-CORR Analyzer
operation, and Yulianto for laboratory supports.
REFERENCES
Al-Chaarani GR, Gentzbittel L, Huang XQ, Sarrafi AA. 2004.
Genotypic variation and identification of QTLs for agronomic
traits, using AFLP, and SSR markers in RILs of sunflower
(Helianthus anuus L.) Theor Appl Genet 109:1353-1360.
Chang ST. 1999. World production of cultivated edible and
medicinal mushrooms in 1997 with emphasis on Lantinus
edodes (Berk) Sing. in China. Inter J Med Mushrooms 1:291300.
Eger G. 1978. Rapid method for breeding Pleurotus ostreatus.
Mushroom Sci 9:567-576.
Mardia KV, Kent JT, Bibby JM. 1979. Multivariate Analysis.
London: Acad Pr.
Ming R, Kim MS, Moore PH, Zee P, Fitch MM, Steiger DL,
Manshardt RM, Raull RE, Drew RA, Sekioka T. 2002. Genetic
diversity of Carica papaya as revealed by AFLP markers.
Genome 45:503-512.
Nei M. 1973. Analysis of gene diversity in subdivided population.
Proc Natl Acad Sci USA 70:3321-3323.
Park YH, West MAL, St Clair DA. 2004. Evaluation of AFLP for
germplasm fingerprinting and assessment of genetic diversity
in cultivar of tomato (Lycopersicon esculentum L.). Genome
47:510-518.
Parlindungan AK. 2003. Karakteristik pertumbuhan dan produksi
jamur tiram putih (Pleurotus ostreatus) dan jamur tiram kelabu
(Pleurotus sajor Caju) pada baglog alang-alang. J Nat Indon
5:152-156.
Raeder U, Broda P. 1985. Rapid preparation of DNA from
filamentous fungi. Lett in Appl Microbiol 1:17-20.

26

JUSUF

Sambrook J, Fritsh EP, Maniatis T. 1989. Molecular Cloning. A
Laboratory Manual. Second Edition. Cold Spring Harbour Lab Pr.
Velazquez-Cedeno MA, Farnet AM, Ferre E. 2004. Variation of
lignocellulosic in dual cultures of Pleurotus ostreatus and
Trichoderma longibrachitum on unstrerilized wheat straw.
Mycologia 96:712-719.
Volk GM, Henk AD, Rihards CM. 2004 Genetic diversity among
US garlic clones as detected using AFLP methods. J Amer Soc
Hort Sci 129:559-569.

HAYATI J Biosci
Vos P, Hogers R, Bleeker M, Reijans M, van de Lee, Hornes M,
Frijters A, Pot J, Peleman J, Kuiper M, Zabeau M. 1995.
AFLP: a new technique for DNA finggerprinting. Nucleic Acids
Res 23:4407-4414.
Widiastuti H, Suharyanto, Wulaningtyas A, Sutamihardja. 2008.
Activity of lignolytic enzymes during growth and fruiting
bodies development of white rot fungi Omphalina sp. and
Pleurotus ostreatus. Hayati 15:140-144.